We detected hepatitis C virus (HCV) RNA in the semen of one third of HCV
viraemic men. Seminal viral loads were low, but the semen could be

infectious and the role of sexual transmission in the spread of HCV infection
should not be underestimated. Sexual transmission of hepatitis C virus (HCV) is
still debated and although two isolated cases of HCV detection in semen have
been reported, findings from extensive studies of 70 viraemic patients could

not confirm this result.To study the presence of HCV in semen, we developed a
reverse transcriptase PCR method that was highly sensitive and adapted to
detecting the virus in semen samples. We looked for HCV-RNA in semen samples
from 21 untreated HCV viraemic men. Among them 15 were HIV-positive patients
treated with antiretroviral therapy. Paired blood and semen samples were
obtained.

HCV-RNA detection in blood samples was done with Cobas Amplicor 2.0

(Roche, Meylan, France) with a sensitivity of 100 copies/mL, and quantification
of blood viral load was obtained using Amplicor HCV Monitor (Roche) with a
sensitivity of 1000 copies/mL. HCV genotypes were determined by INNO-LIPA
HCV II (Innogenetics, Rungis, France). Semen was fractionated with a
Percoll gradient (Sigma-Aldrich, Saint Quentin Fallavier, France) and seminal
fluid was frozen at 280†C. Seminal plasma was tested either pure or
prediluted 1:1 in a 5 g/L bromelain solution (Sigma-Aldrich). HCV-RNAs were
extracted from seminal plasma with Nuclisens kit (Organon Teknica, Fresnel,
France) and internal controls

were added to validate the extraction and amplification steps. Amplification of
extracted RNA was done with Cobas Amplicor 2.0 and quantification with the
Amplicor HCV monitor. Seminal plasma extractions were done on large
volumes (300 to 1000 mL) and extracted RNA was amplified at a high
concentration. These modifications of the procedure allowed us to obtain a
sensitivity of 20 and 100 copies per mL of seminal fluid for qualitative and
quantitative PCR, respectively.

Statistical analysis was done using Mann-Whitney analysis. HCV RNA was detected
in the blood of all patients, plasma viral load and genotype were obtained
for 18 patients, with a median viral load of 5…47 log/mL [2…27-6…30],
and all genotypes (1 to 4) were represented (table). Eight seminal plasma
samples of 21 (38%) were found to contain HCV-RNA (table). The median blood
viral load in patients with positive semen was higher than in patients
with negative semen, but the difference was not significant (figure). Semen
viral loads were low. In three samples 172,

149, and 134 copies/mL were detected; in the other five cases HCV-RNA detection
was negative using the quantitative method although it was positive with the
qualitative PCR and therefore the viral loads could be estimated between
20 and 100 copies/mL.

*Genotypes were available for 18 patients out of the 21, and for seven of eight
patients presenting with a positive semen. Number of patients with a positive
semen.

AUTHORS COMMENTS

The discrepancy between such a high recovery of positive semen and the negative
results of previous studies can be explained by the differences in the method
used for HCV detection in semen. First, we were able to overcome PCR inhibition
by using both Nuclisens extraction and dilution of samples in bromelain. The
presence of PCR inhibitors has been a major hindrance in previo us studies, with
PCR inhibition identified in more than half of the semen samples.4 We used
bromelain (a non-specific cysteine protease) to efficiently liquefy semen
samples, to overcome PCR inhibitors. Indeed, internal control OD values were
significantly higher (mean OD value=3…018) in diluted semen (n=17) than in
undiluted semen (mean OD value=1…381, n=15; p=0…013). Second, we used a
reverse transcriptase PCR, which is more sensitive than those used in previous
studies, with a sensitivity of 20 copies per mL of seminal fluid compared with
600 copies/mL.4 The high sensitivity of the method used was critical since HCV
viral loads in semen were less than 600 copies/mL.

AUTHOR'S CONCLUSIONS

The presence of HCV-RNA in semen is a strong argument in favour of HCV sexual
transmission from men to women. HCV viral loads detected in semen were low,
which suggests that the risk of HCV sexual transmission is probably also low. We
are aware that only further studies using experimental infection in a cell
culture system or an animal model would prove that HCV-RNA positivity in semen
reflects the presence of infectious virus. However, our results are consistent
with epidemiological data which suggested that HCV sexual transmission may occur
with a low risk.5 Finally, these results should be considered in the broader
perspective of safety in laboratories and for counselling and management of
HCV-serodifferent couples who intend to embark on medically assisted
reproduction.